Hyaluronic acid is an important polysaccharide consisting of two repeating units of β-(1,3)-D-glucuronic acid and β-(1,4)-N-acetyl-D-glucosamine. It is characterized by a high molecular weight ranging from 5.104 to 5.106 g·mol−1 which depends on the isolation method and on the initial material used. Hyaluronic acid, and particularly its sodium salt known as hyaluronan, is and essential constituent of connective tissues and of synovial joint fluid. Moreover it plays a significant role in numerous biological processes, such as hydration, organization of proteoglycans, cellular differentiation, proliferation and angiogenesis. This polysaccharide, which is strongly hydrophilic, is water soluble in the form of salts within the entire pH scale.
Carrier Systems on the Basis of Hyaluronic Acid
Due to the hydrophilic nature of its native form, hyaluronic acid cannot serve as an effective carrier for hydrophobic substances. For this reason, hydrophobic functional groups have to be linked to a polymeric chain of hyaluronic acid. In case that such hydrophobic functional groups have sufficient quantities and lengths, an auto-aggregation process involving the same can be initiated resulting in the formation of hydrophobic domains within the structure of hyaluronan. Afterwards, small molecules of water insoluble substances can be linked to such domains by means of non-covalent bonds. The resulting structure is often referred to as polymeric nanomicelle in the literature. wherein the core of the micelle is hydrophobic, thus enabling the dissolution of small nonpolar molecules to take place, while the shell of the same is hydrophilic, thus enabling the polymeric micelle itself to be dissolved in an aqueous environment. A polymeric micelle, which does not exceed 200 nm in size (diameter), can be referred to as nanomicelle.
Carrier systems formed by polymeric micelles on the basis of modified hyaluronic acid are known from conjugates of hyaluronan with alkylamines (Liu et al., 2011) and folic acid. Nevertheless, the presence of highly toxic and teratogenic formamide is deemed to be essential for the preparation of the above hyaluronan derivative. A similar method of preparation of polymeric micelles was employed for obtaining redox-sensitive micelles (Li et al., 2011). It is obvious that such micellar systems are not usable in biological applications due to the presence of highly toxic reagents.
The conjugation of hyaluronic acid with other polymers (such as those of lactic or glycolic acids) through the bond mediated by the carboxylic group of D-glucuronic acid and possibly through the incorporation of low-molecular substances is claimed in the U.S. Pat. No. 7,767,806 wherein the authors mention the biocompatibility of the polymer which, however, has been neither described nor proved by tests. In another case, the low-molecular hyaluronan (9-45 kDa) was covalently modified (in the position of the carboxylic group of glucuronic acid) by hydrophobic amines having various chain lengths and by positively charged spermine used as a cationic segment (Shen. Li, Tu & Zhu, 2009). The purpose of the latter was to prepare a carrier for genes. However, the use of spermine for the above purpose is restricted due to its acute and subacute toxicity (Til, Falke, Prinsen & Willems, 1997). The critical micellar concentration of the formed polymeric micelles having 125-555 in diameter was determined to be higher than 0.04 mg·mL−1. In addition to the fact that the value of the critical micellar concentration is too high and hence does not enable any extreme dilution of micellar system to be achieved (e.g. in the bloodstream), the micelles having the above mentioned size are not considered to be suitable candidates for the passive distribution of medicines within the human body because the size of a polymeric micelle is determinative for the ability of the same to reach a tumor location or infarct lesion through a disrupted venous wall. In such cases, polymeric micelles having 20-100 nm in diameter are preferred (Wang. Mongayt & Torchilin, 2005).
The modification of the carboxylic group of glucuronic acid was utilized for the preparation of polymeric hyaluronan micelles on the basis of the electrostatic interaction between negatively charged hyaluronic acid and positively charged styryl pyridinium (Tao, Xu, Chen, Bai & Liu, 2012). However, the use of such polymeric micelles in vivo is restricted due to the fact that the interactions of styryl pyridinium with nerve terminals and muscarine receptors has not been sufficiently clarified so far, although such interactions play an important role in connection with the regulation of releasing neurotransmitters from neurons (Mazzone, Mori, Burman, Palovich, Belmonte & Canning, 2006).
In the patent documents U.S. 7,993,678 and WO 2007/033677, the method of preparation alkyl/aryl-succinic derivatives of hyaluronan is claimed, such derivatives being also usable for the encapsulation of active nonpolar substances. In the latter case, the modification involves the primary hydroxyl groups of hyaluronan while the carboxylic group remains unchanged. A disadvantage of the above modification reaction is the alkaline pH range (pH 8.5-9.0) in which the reaction of cyclic anhydrides with hyaluronan takes place. In fact, such alkaline pH values can initiate the hydrolysis of anhydrides and, hence, cause the efficacy of the modification process to decrease. This would be particularly considerable in an industrial scale. In the alkyl/aryl-succinic derivatives of hyaluronan, which had been prepared in the above manner and had the substitution degree of 44%, the ability to form micelles (to aggregate) in an aqueous environment was proves when the respective concentration were higher than 0.003-0.004 mg·mL−1. The observed size of the polymeric micelles ranged between 50 and 200 nm. The disadvantage of such derivatives, however, consists in the increase of the total negative charge of hyaluronan caused by the presence of an additional COO− group in the modifying alkyl/aryl-succinic functional group. The negative charge of the molecule may have a significant unfavourable influence on the interaction between cells and the respective carrier system (Wang, Mongayt & Torchilin, 2005). One of the limiting factors for the injection application of the derivatives prepared in the above manner consists in their low solubility (Eenschooten, Guillaumie, Kontogeorgis, Stenby & Schwach-Abdellaoui, 2010). Another disadvantage of such derivatives consists in the instability of the ester bonds during thermal sterilization processes, such as autoclaving ones. In the patent documents U.S. Pat. No. 7,993,678 and WO 2007/033677, merely the method of direct dissolution of nonpolar substances in the solutions of alkyl/aryl-succinic derivatives of hyaluronan, including the formation of a stable emulsion, is described. The main disadvantage of the direct method of encapsulation of nonpolar substances consists in the low resulting bonding capacities of polymeric micelles (Kedar, Phutane, Shidhaye & Kadam, 2010). Although the above patents claim the utilization of the structure of modified hyaluronan for carrier systems, they do not provide any further possible way of linking a hydrophobic substance to the given structure of hyaluronan in addition to the emulsion system. For this reason, the provision both a polymeric carrier system having a sufficient bonding capacity is completely missing which would be one of the basic characteristics required for a realistic assessment of applicability. Moreover, no details relating to cytotoxicity and cellular interactions are mentioned and hence it is not possible to a conclusion about whether the claimed structure is actually applicable for an active transfer of hydrophobic substances into cells, which conclusion is essential in pharmaceutical applications.
In a further publication (Šmejkalová, Hermannová, Šuláková, Pršová, Kučerík & Velebný, 2012), hydrophobic domains of hyaluronan are described which originate from the aggregation of C6-acyl chains linked to hyaluronan by covalent bonds. The derivatives described in the latter publication, however, are not entirely free from residual solvents. Besides that, neither the formation nor the characterization of polymeric micelles is discussed in the above publication. Moreover, the symmetrical anhydrides mentioned in the above publication are not usable for the formation of bonds between long alkyl chains. Carboxylic acids having long aliphatic chains are very expensive and, on top of that, at least one mole of acid gets lost during the preparation of one mole of the final reagent. The publication does not discuss the cytotoxicity of the prepared derivatives, either.
The preparation of butyric esters of polysaccharides including a corresponding pharmaceutical composition is claimed in the patent EP 0941253. The claimed methodology of preparation, however, enables only very low degrees of substitution to be achieved (3% max.). The quantity of the hydrophobic substance. which is linked to the prepared derivatives by means of a non-covalent bond, is unfavourably influenced by such a low degree of substitution. The butyric esters of hyaluronan were further prepared in accordance with the patent WO 2005/092929 wherein, however, non-aqueous conditions. Consequently, the transformation of hyaluronan into a quaternary ammonium salt may be accompanied by the degradation of hyaluronan. The achieved degree of substitution is lower than 0.1% and therefore such ester derivatives are not suitable for the preparation of carrier systems. Similar results were obtained when the simultaneous esterification of hyaluronan with the anhydride of butyric acid and the chloride of retinoic acid was carried out (WO 2004/056877).
A composition of polymeric micelles on the basis of modified hyaluronan (HA)-[O(C═O)NH-M]p, wherein M represents a modifying unit comprising the alkyl functional group C2-16 and p represents a multiple of 3-4, and pharmaceutically active molecules is claimed by the patents U.S. 2010/0316682 and EP1538166A1. The main drawback of such derivatives consists in that dibutyltin laurate, which is known as a substance having an immunotoxic and teratogenic potential, is used for performing the modification of hyaluronan. The latter substance is mostly used in the modification processes related to the production of adhesives and is listed by the European Environment Agency due to its acute toxicity (Boyer, 1989). Another drawback of the claimed derivatives consists in their conjugation with polymers, e.g. with polyethylene glycol, which are extraneous in relation to the human body and can cause inflammatory reactions or give rise cytotoxic degradation products when used for intravenous or topical applications. Moreover, repeated application of polymeric micelles, where polyethylene glycol formed the hydrophilic segment, led to accelerated elimination of those micelles from the bloodstream due to the formation of anti-PEG IgM antibodies (Gong, Chen, Zheng. Wang & Wang, 2012).
Paclitaxel was successfully incorporated into the micelles of modified hyaluronan by means of poly lactic-co-glycolic acid (PLGA) (Kim, Lee, Jang & Park, 2009). The incorporation was carried out with the use of a dialythic method wherein both the polymer and the respective bound substance were dissolved in DMSO and the resulting solution was dialyzed against H2O. In the latter case, the bonding capacity of prepared polymeric micelles of 4.5% by weight was obtained. The main disadvantage of such carrier systems consists in the presence of PLGA polymers, which are extraneous with respect to the human body and may not represent a fully biodegradable system. Another disadvantage consists in the presence of residual DMSO in the final products.
Modification of Hyaluronan with Long-Chain Carboxylic Acids
The modification of polysaccharides with carboxylic acids mostly requires a commercially available anhydride of the given acid (WO 1996/035720. WO 2007/033677. (Šmejkalová, Hermannová, Šuláková, Pršová, Kučerík & Velebný, 2012), EP 0893451). The main disadvantages of such commercially available anhydrides consist in their susceptibility to hydrolysis and in the possible presence of impurities. Moreover, anhydrides of some acids (e.g. undecane-carboxylic acids) are not commercially available. Some of the available anhydrides are very expensive (e.g. those of oleic, linoleic or linolenic acids). Thus, the unavailability, high price and instability of such anhydrides make the large-scale preparation of modified polysaccharides difficult.
Acid anhydrides can be substituted with other acid derivatives which are usable for the esterification of hyaluronan. The patent WO 2010/105582 claim the method of activation of carboxylic acids by means of ethylchloroformiate in non-aqueous conditions, wherein O-acyl-O′-alkyl carbonates are formed which are subsequently usable for the esterification of hyaluronan. The disadvantage of such activation consists in the formation of toxic and potentially explosive gases. A similar method of activation with ethylchloroformiate is disclosed in the patents U.S. Pat. No. 3,720,662 and CZ 20060605.
Another known method is based on the esterification of polysaccharides with carboxylic acids under presence of imidazole (U.S. 2012/0172587). However, the claimed method of preparation requires high reaction temperatures (90-200° C.) which are not applicable to hyaluronan due its degradation under elevated temperatures.
The European patent EP 0893451 claims the esterification of polysaccharides with anhydrides carboxylic acids by means of the method of supercritical extraction. The disadvantages of the latter esterification procedure consist in the necessary high pressure and in the high equipment cost.
For the above reasons, it is very important to find an alternative method of activation of long-chain carboxylic acids, which method would be an in-situ applicable one. One of the possible technical solutions is based on the activation of carboxylic acids with a derivative of 2,4,6-trichlorobenzoic acid accompanied by the formation of an anhydride. For the first time, an anhydride of 2,4,6-trichlorobenzoic acid was used in combination with the DMAP catalyst for a rapid esterification of macrocyclic substances under moderate reaction conditions (Inanaga, Hirata, Saeki, Katsuki & Yamaguchi, 1979). This method of esterification. however, has not yet been applied to the modification of polysaccharides, particularly of hyaluronic acid, because of the possible exothermic reaction accompanied with the degradation of the respective polysaccharide.